Development of a mature biofilm on the tooth surface is an important component of bacterial survival and pathogenesis in the oral cavity. Although over the past years, the complexity of the oral biofilm has been rapidly revealed, understanding how bacteria build up mature pathogenic biofilms in a well- defined way, is still lacking, leading to difficulty in identifying and designing new targets for anti-biofilm therapies. Toward this end, the study of the major Streptococcus mutans autolysin AtlA, shown to be required for biofilm maturation, is a new, but critical pathway for a more complete understanding for biofilm development of this organism. The demonstrated ability of the AtlA pathway to modulate cell surface remodeling is a major driving force for the studies outlined in this proposal, providing a new paradigm for the complex biofilm process particularly as related to the cell surface structure and remodeling process. Under this scope, we will focus our efforts in three interrelated areas. In Specific Aim1, we will elucidate how AtlA is functionally and structurally associated with the cell surface through analysis of repeat domains in the AtlA protein. In the Specific Aim 2, we hypothesize that surface localization and activity of AtlA are modulated primarily by the gene products encoded within the atlA operon. In this Aim, we also attempt to identify additional AtlA-associated components, enhancing our understanding of the architecture and function of AtlA on the cell surface. And in the Specific Aim 3, we will perform a pilot experiment in a mouse caries model to begin to analyze the in vivo impact of AtlA in S. mutans caries development. Overall, this application manipulates an as yet uncharacterized role of AtlA in S. mutans biofilm formation, and data derived from these proposed studies have a high potential to facilitate the development of innovative therapeutic strategies to treat dental caries, e.g. by interfering with the autolytic pathways necessary for the proper development of cariogenic biofilms. This project is also part of our long-term objective elucidating the molecular basis for biofilm maturation and its underlying mechanisms.